Preparation of Vaccines Using Photosensitizers and Light

ABSTRACT

Methods are provided for treating a vaccine containing infectious particles which may be viral, bacterial, and/or cellular in nature. Preferred methods include the steps of adding an effective, non-toxic amount of an endogenous photosensitizer to the fluid and exposing the fluid to photoradiation sufficient to inactivate the infectious particles but not enough to damage the antigenic characteristics of the infectious particles.

CROSS REFERENCE To RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/325402, filed Dec. 20, 2002 which is a non-provisional ofProvisional U.S. Patent Application No. 601342,851 filed Dec. 20, 2001;and is a continuation-in-part of U.S. patent application Ser. No.09/586147 filed Jun. 2, 2000 which is a continuation-in-part of U.S.patent application Ser. No. 09/357188 filed Jul. 20, 1999 now issued asU.S. Pat. No. 6,277,337; which is a continuation-in-part of U.S. patentapplication Ser. No. 09/119666 filed Jul. 21, 1998 now issued as U.S.Pat. No. 6,258,577; all of which are incorporated herein in theirentirety to the extent not incompatible herewith.

FIELD OF THE INVENTION

The present invention relates to the preparation of vaccines. Moreparticularly, the invention relates to inactivation of infectiousparticles in a vaccine whether viral, bacterial or cellular in nature.The infectious particles are inactivated using an endogenousphotosensitizer and light, under conditions which limit antigenicdegradation of the infectious particles in order to elicit an immuneresponse but prevent replication of the infectious particles.

BACKGROUND OF THE INVENTION

Every day, the body is bombarded with bacteria, viruses and otherinfectious agents. When a person is infected with a disease-causing orinfectious agent, the body's immune system attempts to mount a defenseagainst it. When the defense is successful, immunity against theinfectious agent results. When the defense is not successful, aninfection may result.

In the process of developing immunity to the infectious agent, the Bcells of infectious agent and create a “memory” of this experience thatcan be called upon for protection when exposed to the same infectiousagent again months or years later. The next time the person encountersthis particular infectious agent, the circulating antibodies quicklyrecognize it and enable it to be eliminated from the body by otherimmune cells before signs of disease develop. It is estimated thatantibodies which can recognize over 10,000 different antigens or foreign(non-self) infectious agents are circulating in the blood stream.

A vaccine works in a similar way in that it produces an immunogenicresponse. However, instead of initially suffering the natural infectionand risking illness in order to develop this protective immunity,vaccines create a similar protective immunity without exposing the bodyto the disease.

Development of vaccines against both bacterial and viral diseases hasbeen one of the major accomplishments in medicine over the past century.While effective vaccines have been developed for a large number ofdiseases, the need for development of safe and effective vaccines for anumber of additional diseases remains.

Several basic strategies are used to make vaccines. One strategy isdirected toward preventing viral diseases by weakening or attenuating avirus so that the virus reproduces very poorly once inside the body.Measles, mumps, rubella (German measles) and chickenpox (varicella)vaccines are made this way. Whereas natural viruses usually causedisease by reproducing themselves many thousands of times, weakenedvaccine viruses reproduce themselves approximately 20 times. Such a lowrate of replication is generally not enough to cause disease. Althoughthe preparation of live, attenuated infectious agents as vaccines willoften provide improved immunologic reactivity, such methods do increasethe risk that the vaccine itself will be the cause of infection, andthat the attenuated organism will propagate and provide a reservoir forfuture infection. One or two doses of live “weakened” viruses mayprovide immunity that is life long; however, such vaccines cannot begiven to people with weakened immune systems.

Another way to make viral vaccines is to inactivate the virus. By thismethod, viruses are completely inactivated or killed using a chemical.Killing the virus makes the virus unable to replicate in a body andcause disease. Polio, hepatitis A, influenza and rabies vaccines aremade this way. The use of inactivated or killed bacterial or viralagents as a vaccine used to induce an immunogenic response, althoughgenerally safe, will not always be effective if the immunogeniccharacteristics of the agent are altered. An inactive virus can be givento people with weakened immune systems, but must be given multiple timesto achieve immunity.

Vaccines may also be made using parts of the virus. With this strategy,a portion of the virus is removed and used as a vaccine. The body isable to recognize the whole virus based on initial exposure to a portionof the virus. The hepatitis B vaccine for example, is composed of aprotein that resides on the surface of the hepatitis B virus.

Vaccines are also made to help combat diseases caused by bacteria.Several bacterial vaccines are made by taking the toxins produced bybacteria and inactivating them using chemicals. By inactivating thetoxins, the bacteria no longer causes disease. Diphtheria, tetanus andpertussis vaccines are made this way. Another strategy to make bacterialvaccines is to use part of the sugar coating (or polysaccharide) of thebacteria to induce the immunogenic response. Protection against certainbacteria are based on responsive immunity to this sugar coating.

Thus, one must generally choose between improved effectiveness orgreater degree of safety when selecting between the inactivation andattenuation techniques for vaccine preparation. The choice isparticularly difficult when the infectious agent is resistant toinactivation and requires highly rigorous inactivation conditions whichare likely to degrade the antigenic characteristics which help to inducean immune response and provide subsequent immunity.

In addition to the dead or weakened infectious agent, vaccines usuallycontain sterile water or saline. Some vaccines are prepared with apreservative or antibiotic to prevent bacterial growth. Vaccines mayalso be prepared with stabilizers to help the vaccine maintain itseffectiveness during storage. Other components may include an adjuvantwhich helps stimulate the production of antibodies against the vaccineto make it more effective.

Methods to prepare vaccines today involve treating samples withglutaraldehyde or formaldehyde to fix or cross-link the cells orinfectious particles. Such treatments generally involve denaturation ofthe native forms of the infectious particles. A disadvantage to thisapproach is that the protein coats of the infectious particles aredamaged by this process, and thus may not be recognized by the immunesystem.

Therefore, the need exists for a method to prepare vaccines that arerecognized by the immune system but do not replicate once inside thebody.

The use of photosensitizers, compounds which absorb light of a definedwavelength and transfer the absorbed energy to an energy acceptor, areknown to be useful in the sterilization of blood components. Forexample, European Patent application 196,515 published Oct. 8, 1986,suggests the use of non-endogenous photosensitizers such as porphyrins,psoralens, acridine, toluidines, flavine (acriflavine hydrochloride),phenothiazine derivatives, and dyes such as neutral red, and methyleneblue, as blood additives. Protoporphyrin, which occurs naturally withinthe body, can be metabolized to form a photosensitizer; however, itsusefulness is limited in that it degrades desired biological activitiesof proteins. Chlorpromazine, is also exemplified as one suchphotosensitizer; however its usefulness is limited by the fact that itshould be removed from any fluid administered to a patient after thedecontamination procedure because it has a sedative effect.

Goodrich, R. P., et al. (1997), “The Design and Development ofSelective, Photoactivated Drugs for Sterilization of Blood Products,”Drugs of the Future 22:159-171 provides a review of somephotosensitizers including psoralens, and some of the issues ofimportance in choosing photosensitizers for decontamination of bloodproducts. The use of texaphyrins for DNA photocleavage is described inU.S. Pat. No. 5,607,924 issued Mar. 4, 1997 and U.S. Pat. No. 5,714,328issued Feb. 3, 1998 to Magda et al. The use of sapphyrins for viraldeactivation is described in U.S. Pat. No. 5,041,078 issued Aug. 20,1991 to Matthews, et al. Inactivation of extracellular enveloped virusesin blood and blood components by Phenthiazin-5-ium dyes plus light isdescribed in U.S. Pat. No. 5,545,516 issued Aug. 13, 1996 to Wagner. Theuse of porphyrins, hematoporphyrins, and merocyanine dyes asphotosensitizing agents for eradicating infectious contaminants such asviruses and protozoa from body tissues such as body fluids is disclosedin U.S. Pat. No. 4,915,683 issued Apr. 10, 1990 and related U.S. Pat.No. 5,304,113 issued Apr. 19, 1994 to Sieber et al. The reactivity ofpsoralen derivatives with viruses has been studied. See, Hearst andThiry (1977) Nuc. Acids Res. 4:1339-1347; and Talib and Banerjee (1982)Virology 118:430-438. U.S. Pat. No.4,124,598 and 4,196,281 to Hearst etal. suggest the use of psoralen derivatives to inactivate RNA viruses,but include no discussion of the suitability of such inactivated virusesas vaccines. U.S. Pat. No. 4,169,204 to Hearst et al. suggests thatpsoralens may provide a means for inactivating viruses for the purposeof vaccine production but presents no experimental support for thisproposition. European patent application 0 066 886 by Kronenberg teachesthe use of psoralen inactivated cells, such as virus-infected mammaliancells, for use as immunological reagents and vaccines. Hanson (1983) in:Medical Virology II, de la Maza and Peterson, eds., Elsevier Biomedical,New York, pp. 45-79, reports studies which have suggested that oxidativephotoreactions between psoralens and proteins may occur. Wiesehahn etal. discloses in U.S. Pat. Nos. 4,693,981 and 5,106,619 the use ofpsoralens to prepare inactivated viral vaccines. These patents disclosepreparing vaccines by treating viruses with furocoumarins and longwavelength UV light for a time period sufficiently long enough to renderthe virus non-infectious but less than that which would result indegradation of its antigenic characteristics under conditions whichlimit the availability of oxygen and other oxidizing species. Swartzdiscloses in U.S. Pat. No. 4,402,318 a method of producing a vaccine byadding methylene blue and exposing the vaccine to light and an electricfield concurrently to completely inactivate the viruses, bacteria, cellsand toxins. Dorner et al. in U.S. Pat. No. 6,165,711 discloses a processfor disintegrating nucleic acids to make vaccines by exposingbiologically active material to phenothiazine and a laser beam.

The mechanism of action of psoralens is described as involvingpreferential binding to domains in lipid bilayers, e.g. on envelopedviruses and some virus-infected cells. Photoexcitation of membrane-boundagent molecules leads to the formation of reactive oxygen species suchas singlet oxygen which causes lipid peroxidation. A problem with theuse of psoralens is that they attack cell membranes of desirablecomponents of fluids to be decontaminated, such as red blood cells, andthe singlet oxygen produced during the reaction also attacks desiredprotein components of fluids being treated.

U.S. Pat. No. 4,727,027 issued Feb. 23, 1988 to Wiesehahn, G. P., et al.discloses the use of furocoumarins including psoralen and derivativesfor decontamination of blood and blood products, but teaches that stepsmust be taken to reduce the availability of dissolved oxygen and otherreactive species in order to inhibit denaturation of biologically activeproteins. Photoinactivation of viral and bacterial blood contaminantsusing halogenated coumarins is described in U.S. Pat. No. 5,516,629issued May 14, 1996 to Park, et al. U.S. Pat. No. 5,587,490 issuedDec.24, 1996 to Goodrich Jr., R. P., et al. and U.S. Pat. No. 5,418,130to Platz, et al. disclose the use of substituted psoralens forinactivation of viral and bacterial blood contaminants. The latterpatent also teaches the necessity of controlling free radical damage toother blood components. U.S. Pat. No. 5,654,443 issued Aug. 5, 1997 toWollowitz et al. teaches new psoralen compositions used forphotodecontamination of blood. U.S. Pat. No. 5,709,991 issued Jan. 20,1998 to Lin et al. teaches the use of psoralen for photodecontaminationof platelet preparations and removal of psoralen afterward. U.S. Pat.No. 5,120,649 issued June 9, 1992 and related U.S. Pat. No. 5,232,844issued Aug. 3, 1993 to Horowitz, et al., also disclose the need for theuse of “quenchers” in combination with photosensitizers which attacklipid membranes, and U.S. Pat. No. 5,360,734 issued Nov. 1, 1994 toChapman et al. also addresses this problem of prevention of damage toother blood components.

Photosensitizers which attack nucleic acids are known to the art. U.S.Pat. No. 5,342,752 issued Aug. 30, 1994 to Platz et al. discloses theuse of compounds based on acridine dyes to reduce parasiticcontamination in blood matter comprising red blood cells, platelets, andblood plasma protein fractions. These materials, although of fairly lowtoxicity, do have some toxicity e.g. to red blood cells. This patentfails to disclose an apparatus for decontaminating blood on aflow-through basis. U.S. Pat. No. 5,798,238 to Goodrich, Jr., et al.,discloses the use of quinolone and quinolone compounds for inactivationof viral and bacterial contaminants.

Binding of DNA with photoactive agents has been exploited in processesto reduce lymphocytic populations in blood as taught in U.S. Pat. No.4,612,007 issued Sep. 16, 1986 and related U.S. Pat. No. 4,683,889issued Aug. 4, 1987 to Edelson.

Riboflavin (7,8-dimethyl-10-ribityl isoalloxazine) has been reported toattack nucleic acids. U.S. Pat. Nos. 6,258,577 and 6,277,337 issued toGoodrich et al. disclose the use of riboflavin and light to inactivatemicroorganisms which may be contained in blood or blood products. U.S.Pat. No. 6,268,120 to Platz et al. discloses riboflavin derivativeswhich may be used to inactivate microorganisms.

All publications referred to herein are hereby incorporated by referenceto the extent not inconsistent herewith.

SUMMARY OF THE INVENTION

It is one aspect of the instant invention to provide improved methodsfor inactivating infectious particles, which methods are capable ofinactivating even the most resistant particles under conditions which donot substantially degrade the antigenic structure of the particles. Inparticular, the inactivated infectious particles should be useful asvaccines and free from adverse side effects at the time ofadministration as well as upon subsequent challenge with the liveinfectious agents upon future exposure.

One method for inactivating infectious particles without substantiallydegrading the antigenic characteristics of the particles, may includethe steps of exposing the infectious particles to an inactivation fluidcontaining at least an endogenous photosensitizer in an amountsufficient to render the particles substantially non-infectious; andexposing the infectious particles and inactivation fluid to light of asufficient wavelength to render the particles substantiallynon-infectious but less than that which would result in degradation ofthe antigenic characteristics of the infectious particles.

The particles are rendered substantially non-infectious by damaging thenucleic acids of the infectious particles, preventing the particles fromreplicating one inside the recipient of the vaccine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the riboflavin absorbance spectrum.

FIG. 2 depicts a correlation of light absorbance and hematocrit observedand predicted for red blood cells, and predicted for platelets.

FIG. 3 depicts photodecomposition over time of riboflavin inanticoagulant Acid Citrate Dextrose (ACD) solution. The solid line withcircles indicates percent of initial riboflavin remaining at 373 nm. Thedotted line with squares indicates percent of initial riboflavinremaining at 447 nm.

FIG. 4 depicts the transmission profile of various plastic cuvettes as afunction of wavelength. The solid line represent a 3.2 mm acryliccuvette. The dotted line (---) represents a 3.2 mm UV acrylic cuvette.The dashed line (- - -) represents a 3.2 mm polystyrene (PS) cuvette,and the crossed line indicates a 3.2 mm polycarbonate (PC) cuvette.

FIG. 5 depicts the light flux required in mW per cm² as a function offlow rate, i.e. the flux required to deliver one joule/cm² to a samplein the cuvette.

FIG. 6 depicts the decontamination assembly of this invention.

FIG. 7 depicts inactivation of bacteria in platelet preparations usingvitamin K5 as the photosensitizer as a function of energy ofirradiation.

FIG. 8 depicts inactivation of bacteria as a function of plateletpreparation and energy of irradiation, using 90% platelets and 10%platelet additive solution (90:10) and 30% platelets with 70% additivesolution (30:70).

FIG. 9 shows the effect on inactivation of virus, bacteriophage andbacteria of adding antioxidants to platelet concentrate.

FIG. 10 shows the inactivation curve for Herpes Simplex type II virus asa function of concentration of photosensitizer at an energy ofirradiation of 20 J/cm² using half ultraviolet and half visible light.

FIG. 11 shows inactivation of S. epidermidis at varying concentrationsof photosensitizer and energies of irradiation.

FIG. 12 shows inactivation of φX174 at varying concentrations ofphotosensitizer and energies of irradiation.

FIG. 13 shows inactivation of S. aureus and φX174 at varying energies ofirradiation using a 50:50 mixture of ultraviolet and visible light. FIG.14 shows inactivation of S. epidermidis and HSV-II at varying energiesof irradiation using a 50:50 mixture of ultraviolet and visible light.

FIG. 15 shows inactivation of HSV2 virus in blood bags agitated andirradiated at varying energy levels.

FIG. 16 compares inactivation results for vaccinia virus in variousfluids using ultraviolet light alone or 50:50 visible and ultravioletlight.

FIG. 17 compares inactivation results with and without sensitizer ofvaccinia virus at varying irradiation times.

FIG. 18 compares inactivation of extracellular HIV-1 at 5 and 50 μM ofphotosensitizer and varying irradiation energies.

FIG. 19 compares inactivation of intracellular HIV-1 at 5 and 50 μM ofphotosensitizer and varying irradiation energies.

FIG. 20 compares inactivation of intracellular HIV-1 at 5 and 50 μM ofphotosensitizer and varying irradiation energies, using p24 antigenlevels.

FIG. 21 shows inactivation of HSV-II at varying irradiation levels usingplatelet concentrate and platelet concentrate in media containingplatelet additive solution with ascorbate.

FIG. 22 shows an embodiment of this invention using a bag to contain thefluid being treated and photosensitizer and a shaker table to agitatethe fluid while exposing to photoradiation from a light source.

DETAILED DESCRIPTION

According to the present invention, vaccines useful for the inoculationof mammalian hosts, including both animals and humans, against infectionare provided. The vaccines may be prepared by inactivation of infectiousparticles in an inactivation medium containing an amount of aninactivating endogenous photosensitizer or endogenous photosensitizerderivative sufficient to inactivate the infectious particles uponsubsequent irradiation._Degradation of the antigenic characteristics ofthe infectious particles are reduced or eliminated by use of anendogenous photosensitizer, and in particular an isoalloxazine orisoalloxazine derivative. Suitable vaccines may be prepared by combiningthe inactivated infectious particles with a physiologically-acceptablecarrier, such as water, saline or an adjuvant, in an appropriate amountto elicit an immune response, e.g., the production of serum neutralizingantibodies, upon subsequent inoculation of the host.

As used herein, the term “inactivation of an infectious particle” or“agent” means substantially preventing the infectious particle or agentfrom replicating, either by killing the particles or otherwiseinterfering with its ability to reproduce, while still maintaining theantigenic characteristics of the particles.

Decontamination methods of this invention using endogenousphotosensitizers or endogenously-based photosensitizer derivatives donot substantially destroy the antigenicity on the surface of theinfectious particles but do substantially destroy the nucleic acids ofthe particles and therefore the replicative ability of the particles. Solong as the infectious particles retain sufficient antigenicdetermination to be useful for their intended purpose of inducingimmunity in a mammal, their biological activities are not considered tobe “substantially destroyed.” Infectious particles or agents which maybe present in a vaccine include viruses (both extracellular andintracellular), bacteria, bacteriophages, fungi, blood-transmittedparasites, protozoa, virus infected cells, cancer cells, dendritic cellsor altered immune cells. Exemplary viruses which may be made intovaccines include acquired immunodeficiency (HIV) virus, hepatitis A, Band C viruses, sinbis virus, cytomegalovirus, vesicular stomatitisvirus, herpes simplex viruses, e.g. types I and II, human T-lymphotropicretroviruses, HTLV-III, lymphadenopathy virus LAV/IDAV, parvovirus,transfusion-transmitted (TT) virus, Epstein-Barr virus, adenovirus,papovaviruses, poxviruses, picornavirus, paramyoxovirus, coronavirus,calicivirus, togavirus, rhabdovirus and others known to the art.

Bacteriophages include φX174, φ6, λ, R17, T₄, and T₂.

Exemplary bacteria may include but not be limited to P. aeruginosa, S.aureus, S. epidermidis, L. monocytogenes, E. coli, K pneumonia, S.marcesoens, E. faecalis, B. subtilis, S. pneumoniae, S. pyogenes, S.viridans, B. cereus, E. aerogenes, Propionabacter, K. pneumoniae, C.perfringes, E. cloacae, P. mirabilis, S. cholerasuis, S. liquifaciens,S. mitis, Y. enterocolitica, P. fluorescens, S. enteritidis, C.freundii.

Exemplary cells which may be used to make vaccines include tumor cells,virus infected cells and immune cells which may include dendritic cellsand T and B cells.

Materials which may be treated by the methods of this invention includeany materials or components of vaccines which are adequately permeableto photoradiation to provide sufficient light to achieve inactivation ofthe infectious particles, or which can be suspended or dissolved influids which have such permeability to photoradiation. Examples of suchmaterials may be the infectious particle and/or any components orcompositions included in vaccine preparations such as adjuvants,antibiotics, preservatives, stabilizers, saline and/or water. Theinfectious particles which may be used in a vaccine may be used in whole(the entire infectious particle) or portions of the particle (such asthe protein coat of a virus) may be used.

The term “biologically active” means capable of effecting a change in aliving organism or component thereof. Similarly, “non-toxic” withrespect to the photosensitizers means low or no toxicity to humans andother mammals, and does not mean non-toxic to the particles beinginactivated. “Substantial destruction” of biological activity means atleast as much destruction as is caused by porphyrin and porphyrinderivatives, metabolites and precursors which are known to have adamaging effect on biologically active proteins and cells of humans andmammals.

Similarly, “substantially non-toxic” means less toxic than porphyrin,porphyrin derivatives, metabolites and precursors.

The photosensitizers useful in this invention include anyphotosensitizers known to the art to be useful for inactivatingmicroorganisms or other infectious particles. A “photosensitizer” isdefined as any compound which absorbs radiation of one or more definedwavelengths and subsequently utilizes the absorbed energy to carry out achemical process. Examples of such photosensitizers include porphyrins,psoralens, dyes such as neutral red, methylene blue, acridine,toluidines, flavine (acriflavine hydrochloride) and phenothiazinederivatives, coumarins, quinolones, quinones, and anthroquinones.Photosensitizers of this invention may include compounds whichpreferentially adsorb to nucleic acids, thus focusing their photodynamiceffect upon microorganisms and viruses with little or no effect uponaccompanying cells or proteins. Other photosensitizers are also usefulin this invention, such as those using singlet oxygen-dependentmechanisms. Most preferred are endogenous photosensitizers. The term“endogenous” means naturally found in a human or mammalian body, eitheras a result of synthesis by the body or because of ingestion as anessential foodstuff (e.g. vitamins) or formation of metabolites and/orbyproducts in vivo. Examples of such endogenous gphotosensitizers arealloxazines such as 7,8-dimethyl-10-ribityl isoalloxazine (riboflavin),7,8,10-trimethylisoalloxazine (lumiflavin), 7,8-dimethylalloxazine(lumichrome), isoalloxazine-adenine dinucleotide (flavine adeninedinucleotide [FAD]), alloxazine mononucleotide (also known as flavinemononucleotide [FMN] and riboflavine-5-phosphate), vitamin Ks, vitaminL, their metabolites and precursors, and napththoquinones, naphthalenes,naphthols and their derivatives having planar molecular conformations.The term “alloxazine” includes isoalloxazines. Endogenously-basedderivative photosensitizers include synthetically derived analogs andhomologs of endogenous photosensitizers which may have or lack lower(1-5) alkyl or halogen substituents of the photosensitizers from whichthey are derived, and which preserve the function and substantialnon-toxicity thereof. When endogenous photosensitizers are used,particularly when such photosensitizers are not inherently toxic or donot yield toxic photoproducts after photoradiation, no removal orpurification step is required after decontamination, and treated productcan be directly administered to a patient by any methods known in theart. Preferred endogenous photosensitizers are:

The method of this invention requires mixing the photosensitizer withthe material to be decontaminated. In this particular application, thepreferred material or fluid is one containing infectious particles fromwhich it is desired to make a vaccine. Mixing may be done by simplyadding the photosensitizer or a solution containing the photosensitizerto a fluid to be decontaminated. In one embodiment, the material to bedecontaminated to which photosensitizer has been added is flowed past aphotoradiation source, and the flow of the material generally providessufficient turbulence to distribute the photosensitizer throughout thefluid to be decontaminated. A mixing step may optionally be added. Inanother embodiment, the fluid and photosensitizer are placed in aphotopermeable container and irradiated in batch mode, preferably whileagitating the container to fully distribute the photosensitizer andexpose all the fluid to the radiation.

The amount of photosensitizer to be mixed with the fluid will be anamount sufficient to adequately inactivate the reproductive ability ofan infectious particle, but in many embodiments, still maintain theantigenic properties necessary to induce an immune reaction in a mammaland produce subsequent immunity. As taught herein, optimalconcentrations for desired photosensitizers may be readily determined bythose skilled in the art without undue experimentation. Preferably thephotosensitizer is used in a concentration of at least about 1 μM up tothe solubility of the photosensitizer in the fluid, and preferably about10 μM. For 7,8-dimethyl-10-ribityl isoalloxazine a concentration rangebetween about 1 μM and about 160 μM is preferred, preferably about 10μM.

The fluid containing the photosensitizer is exposed to photoradiation ofthe appropriate wavelength to activate the photosensitizer, using anamount of photoradiation sufficient to activate the photosensitizer asdescribed above, but less than that which would cause damage to theantigenic determinants of the infectious particles and render theinactivated infectious particle unable to induce an immune response in amammal. The wavelength used will depend on the photosensitizer selected,as is known to the art or readily determinable without undueexperimentation following the teachings hereof. Preferably the lightsource is a fluorescent or luminescent source providing light of about300 nm to about 700 nm, and more preferably about 320 nm to about 447 nmof radiation. Wavelengths in the ultraviolet to visible range are usefulin this invention. The light source or sources may provide light in thevisible range, light in the ultraviolet range, or may be a mixture oflight in the visible and ultraviolet ranges.

The activated photosensitizer is capable of inactivating the infectiousparticles present, such as by interfering to prevent their replication.Specificity of action of the photosensitizer is conferred by the closeproximity of the photosensitizer to the nucleic acid of the particle andthis may result from binding of the photosensitizer to the nucleic acid.“Nucleic acid” includes ribonucleic acid (RNA) and deoxyribonucleic acid(DNA). Other photosensitizers may act by binding to cell membranes or byother mechanisms. The photosensitizer may also be targeted to theparticles to be inactivated by covalently coupling to an antibody,preferably a specific monoclonal antibody to the particle.

The fluid containing the photosensitizer may be flowed into aphotopermeable container for irradiation. The term “container” refers toa closed or open space, which may be made of rigid or flexible material,e.g., may be a bag or box or trough. It may be closed or open at the topand may have openings at both ends, e.g., may be a tube or tubing, toallow for flow-through of fluid therein. A cuvette has been used toexemplify one embodiment of the invention involving a flow-throughsystem. Collection bags, such as those used with the Trima™ and Spectra™apheresis systems of GambroBCT, Inc., have been used to exemplifyanother embodiment involving batch-wise treatment of the fluid.

The term “photopermeable” means the material of the container isadequately transparent to photoradiation of the proper wavelength foractivating the photosensitizer. In the flow-through system, thecontainer has a depth (dimension measured in the direction of theradiation from the photoradiation source) sufficient to allowphotoradiation to adequately penetrate the container to contactphotosensitizer molecules at all distances from the light source andensure inactivation of infectious particles in the fluid to bedecontaminated, and a length (dimension in the direction of fluid flow)sufficient to ensure a sufficient exposure time of the fluid to thephotoradiation. The materials for making such containers, depths andlengths of containers may be easily determined by those skilled in theart without undue experimentation following the teachings hereof, andtogether with the flow rate of fluid through the container, theintensity of the photoradiation and the absorptivities of the fluidcomponents, will determine the amount of time the fluid needs to beexposed to photoradiation. For 7,8-dimethyl-10-ribityl isoalloxazine, apreferred amount of radiation is between about 1 J/cm² to 1 20J/cm².

In another embodiment involving batch-wise treatment, the fluid to betreated is placed in a photopermeable container which is agitated andexposed to photoradiation for a time sufficient to substantiallyinactivate the infectious particles, but not enough to destroy theantigenicity of the particles. The photopermeable container ispreferably a blood bag made of transparent or semitransparent plastic,and the agitating means is preferably a shaker table. Thephotosensitizer may be added to the container in powdered or liquid formand the container agitated to mix the photosensitizer with the fluid andto adequately expose all the fluid to the photoradiation to ensureinactivation of the particles.

Photosensitizer may be added to or flowed into the photopermeablecontainer containing the infectious particles to be inactivated. In oneembodiment, the photosensitizer is added to the fluid which is used tosuspend the inactivated infectious particles to create the vaccine. Inanother embodiment, the photosensitizer may be added to the infectiousparticles to be inactivated and the suspension fluid or carrier fluid.

Enhancers may also be added to the fluid to make the process moreefficient and selective. Such enhancers include antioxidants or otheragents to prevent damage to desired fluid components or to improve therate of inactivation of infectious particles and are exemplified byadenine, histidine, cysteine, tyrosine, tryptophan, ascorbate,N-acetyl-L-cysteine, propyl gallate, glutathione,mercaptopropionylglycine, dithiothreotol, nicotinamide, BHT, BHA,lysine, serine, methionine, glucose, mannitol, trolox, glycerol, andmixtures thereof.

This invention may also comprise fluids comprising biologically activeprotein, which may be used to produce passive immunity in a patient.Passive immunity involves administration of antibodies such asimmunoglobins which may be injected into patients to provide short-termprotection to those individuals who have been or will be exposed to aspecific pathogen and is typically used in immunocompromised patientswho are unable to produce an effective immune response with activeimmunization. Fluids containing biologically active proteins and alsocontaining endogenous photosensitizer, endogenously-based derivativephotosensitizer, or photoproduct thereof may be injected into a patientto provide such passive immunity. The fluid may also contain inactivatedmicroorganisms.

In decontamination systems of this invention, the photoradiation sourcemay be connected to the photopermeable container for the fluid by meansof a light guide such as a light channel or fiber optic tube whichprevents scattering of the light between the source and the containerfor the fluid, and more importantly, prevents substantial heating of thefluid within the container. Direct exposure to the light source mayraise temperatures as much as 10 to 15° C., especially when the amountof fluid exposed to the light is small, which can cause denaturizationof blood components. Use of the light guide keeps any heating to lessthan about 2° C. The method may also include the use of temperaturesensors and cooling mechanisms where necessary to keep the temperaturebelow temperatures at which desired proteins in the fluid are damaged.Preferably, the temperature is kept between about 0° C. and about 45°C., more preferably between about 4° C. and about 37° C., and mostpreferably about 22° C.

The photoradiation source may be a simple lamp or may consist ofmultiple lamps radiating at differing wavelengths. The photoradiationsource should be capable of delivering from about 1 to at least about120 J/cm². The use of mixed ultraviolet and visible light is especiallypreferred when the photosensitizer is one which loses its capacity toabsorb visible light after a period of exposure, such as7,8-dimethyl-10-ribityl-isoalloxazine.

Any means for adding the photosensitizer to the fluid to bedecontaminated and for placing the fluid in the photopermeable containerknown to the art may be used, such means typically including flowconduits, ports, reservoirs, valves, and the like.

For endogenous photosensitizers and derivatives having sugar moieties,the pH of the solution is preferably kept low enough, as is known to theart, to prevent detachment of the sugar moiety. Preferably thephotosensitizer is added to the fluid to be decontaminated in apre-mixed aqueous solution, e.g., in water, storage buffer or suspensionsolution.

The photopermeable container for the flow-through system may be atransparent cuvette made of polycarbonate, glass, quartz, polystyrene,polyvinyl chloride, polyolefin, or other transparent material. Thecuvette may be enclosed in a radiation chamber having mirrored walls. Aphotoradiation enhancer such as a second photoradiation source orreflective surface may be placed adjacent to the cuvette to increase theamount of photoradiation contacting the fluid within the cuvette. Thesystem preferably includes a pump for adjusting the flow rate of thefluid to desired levels to ensure substantial decontamination asdescribed above. The cuvette has a length, coordinated with the flowrate therethrough, sufficient to expose fluid therein to sufficientphotoradiation to effect substantial decontamination thereof.

Also preferably the cuvette is spaced apart from the light source asufficient distance that heating of the fluid in the cuvette does notoccur, and light is transmitted from the light source to the cuvette bymeans of a light guide.

Decontamination systems as described above may be designed asstand-alone units or may be easily incorporated into existingapparatuses known to the art for inactivating infectious particles tomake vaccines.

The use of endogenous photosensitizers and endogenously-based derivativephotosensitizers to inactivate infectious particles in vaccines asdisclosed herein is described with reference to the inactivation ofmicroorganisms in blood, separated blood components and other cellularcomponents.

Solutions for suspension of the inactivated infectious particlescomprising endogenous photosensitizers and endogenously-based derivativephotosensitizers as described above are also provided herein. Suchsuspension or additive solutions may contain physiological salinesolution, water, antibiotics, preservatives, stabilizers and adjuvants.The pH of such solutions is preferably between about 7.0 and 7.4. Thesesolutions are useful as carriers for inactivated infectious particles toallow maintenance of quality and viability of the inactivated infectiousparticles during storage. The photosensitizer may be present in suchsolutions at any desired concentration from about 1 μM to the solubilityof the photosensitizer in the solution, and preferably between about 10μM and about 100 μM, more preferably about 10 μM. In a preferredembodiment, the suspension solution also comprises enhancers asdescribed above.

The present invention is suitable for producing vaccines to a widevariety of viruses, including human viruses and animal viruses, such ascanine, feline, bovine, porcine, equine, and ovine viruses. The methodis suitable for inactivating double stranded DNA viruses,single-stranded DNA viruses, double-stranded RNA viruses, andsingle-stranded RNA viruses, including both enveloped and non-envelopedviruses. The following list contains some representative viruses whichmay be inactivated by the method of the present invention. Viruses whichmay be inactivated Representative Viruses dsDNA Adenoviruses Adenovirus,canine adenovirus 2 Herpesviruses Herpes simplex viruses, Feline HerpesI Papovaviruses Polyoma, Papilloma Poxviruses, Vaccinia ssDNA ParvovirusCanine parvovirus, Feline panleukopenia dsRNA Orbiviruses Bluetonguevirus Reoviruses Reovirus ssRNA Calicivirus Feline calicivirusCoronavirus Feline infectious peritonitis Myxovirus Influenza virusParamyxovirus Measles virus, Mumps virus, Newcastle disease virus,Canine distempter virus, Canine parainfluenza 2 virus Picornavirus Poliovirus, Foot and mouth disease virus Retrovirus Feline leukemia virus,Human T-cell lymphoma virus, types I, II and III Rhabdovirus Vesicularstomatitis virus, rabies Togavirus Yellow fever virus, Sindbis virus,Encephalitis virus

Of particular interest are those viruses for which conventional vaccineapproaches have been unsuccessful or marginally successful. For suchviruses, inactivation procedures which are sufficiently rigorous toassure the total loss of infectivity often result in partial or completedestruction of the antigenic characteristics of the virus. With suchloss of antigenic characteristics, the viruses are incapable ofeliciting a protective immunity when administered to a susceptible host.While it would be possible to utilize less rigorous inactivationconditions in order to preserve the antigenic integrity of the virus,this approach is not desirable since it can result in incompleteinactivation of the virus, and increase the potential threat ofinfection of the recipient by the incompletely inactivated virus.

The methods of this invention may also be used to produce vaccines frombacteria or portions of bacteria. Some bacteria which may be used toproduce vaccines include but are not limited to diphtheria, tetanus,pertussis, haemophilus influenzae B, pneumococcus, vibrio cholerae,salmonella typhi and neisseria meningiditis.

Vaccines may be made to treat insect-borne diseases including but notlimited to typhus, malaria, dengue fever, yellow fever, chagus babesin,rickettsia and west nile virus using the methods of this invention.

Vaccines and methods to produce vaccines from inactivated cancer cellsare also contemplated by this invention. For example, vaccines may bemade of autologous tumor cells to elicit a long-term anti-tumor immuneresponse, known as active specific immunotherapy. A tumor may besurgically resected from a patient and the tumor cells purified usingany method known in the art. One such method to prepare autologous tumorvaccines which may be used in this invention is described in Hanna etal. (Hanna M G Jr, Brandhorst J S, Peters L C, Specific immunotherapy ofestablished visceral micrometastases by BCG-tumour cell vaccine alone oras an adjunct to surgery. Cancer 1978; 42: 2613-25) and Peters et al.(Peters L C, Brandhorst J S, Hanna M G Jr., Preparation ofimmunotherapeutic autologous vaccines form solid tumors. Cancer Res1979; 39: 1353-60) which are both herein incorporated by reference intheir entireties to the amount not inconsistent herewith. The purifiedtumor cells may then be treated with an amount of endogenousphotosensitizer and light sufficient to prevent the nucleic acid of thetumor cells from replicating but not enough to damage the antigenicdeterminants on the surface of the tumor cells. Once the tumor cellshave been inactivated, they may be immediately injected back into thepatient, or may be frozen for future use.

One method which may be used to make a vaccine for preventing thereplication of live tumor cells in a mammal include the removal of atumor from a mammal having a tumor; purifying at least some of the tumorcells from the tumor; inactivating the tumor cells by exposing the tumorcells to an endogenous photosensitizer and light at a sufficientwavelength to prevent replication of the tumor cells but notsubstantially destroy the antigenic determinants of the tumor cells; andsuspending the inactivated tumor cells in a suspension solution. Thephotosensitizer may include riboflavin, lumiflavin, lumichrome,napthquinones, napthels and napthalenes, but any photosensitizer whichprevents the replication of the tumor cells but does not substantiallydestroy the antigenic determinants of the cells may be used. Thesuspension solution may be any solution known in the art.

To inactivate infectious particles in a vaccine, a photosensitizer maybe added to the infectious particles and exposed to light of anappropriate wavelength to inactivate the infectious particles whileretaining the antigenic properties of the particles before suspension ina sterile physiologically acceptable medium. Alternatively, aphotosensitizer may be added after the addition of the medium. Theinfectious particles are irradiated after the addition of thephotosensitizer.

The inactivated infectious particles may be formulated in a variety ofways for use as a vaccine. The concentration of the infectious particlesin the vaccine will be in an amount sufficient to induce the productionof antibodies by the body in order to provide long-term immunity as isknown by those skilled in the art. The vaccine may include cells or maybe cell-free. The vaccine may also contain portions of infectiousparticles. The inactivated infectious particles may be resuspended in aninert physiologically acceptable medium, such as ionized water,phosphate-buffered saline, saline, or the like, or may be administeredin combination with a physiologically acceptable immunologic adjuvant,including but not limited to mineral oils, vegetable oils, mineral saltssuch as aluminum and immunopotentiators, such as muramyl dipeptide. Astabilizer may also be added to help the vaccine maintain itseffectiveness during storage. The vaccine may be administeredsubcutaneously, intramuscularly, intraperitoneally, orally, or nasally.Usually, a total specific dosage at a specific site will range fromabout 0.1 ml to 4 ml, where the total dosage will range from about 0.5ml to 8 ml. The number of injections and their temporal spacing may behighly variable, and will depend on the effectiveness of the vaccine andhow well the recipients' immune system responds to the vaccine.

It should be noted that any contaminating microorganisms in apre-existing vaccine made by any known methods may be inactivated astaught by the methods of this invention.

The following examples are offered by way of illustration, not by way oflimitation. The below examples show that blood cells contaminated withpathogens which have been treated by the methods of this inventionretain their biological activity while the amount of pathogens aresubstantially reduced. After treatment with photosensitizer and light,infectious particles to be used as a vaccine retain their antigenicitybut are unable to replicate.

The decontamination method of this invention using endogenousphotosensitizers and endogenously-based derivative photosensitizers isexemplified herein using 7,8-dimethyl-10-ribityl isoalloxazine as thephotosensitizer, however, any photosensitizer may be used which iscapable of being activated by photoradiation to cause inactivation ofinfectious particles. The photosensitizer must be one which does notdestroy desired components of the fluid being decontaminated, and alsopreferably which does not break down as a result of the photoradiationinto products which significantly destroy desired components or havesignificant toxicity. The wavelength at which the photosensitizer isactivated is determined as described herein, using literature sources ordirect measurement. Its solubility in the fluid to be decontaminated orin a combination of carrier fluid and fluid to be contaminated is alsoso determined. The ability of photoradiation at the activatingwavelength to penetrate the fluid to be decontaminated must also bedetermined as taught herein. Appropriate temperatures for the reactionof the photosensitizer with its substrate are determined, as well as theranges of temperature, photoradiation intensity and duration, andphotosensitizer concentration which will optimize microbial inactivationand minimize damage to desired proteins and/or cellular components inthe fluid. Examples 1-7 and FIGS. 1-5 illustrate the determination ofinformation required to develop a flow-through decontamination system ofthis invention.

Once such system requirements have been determined for flow-throughsystems, apparatuses may be designed which provide the correct flowrates, photopermeabilities, and light intensities to cause inactivationof microorganisms present in the fluid, as is taught herein. The fluidto be decontaminated is mixed with photosensitizer and then irradiatedwith a sufficient amount of photoradiation to activate thephotosensitizer to react with microorganisms in the fluid such thatmicroorganisms in the fluid are inactivated. The amount ofphotoradiation reaching microorganisms in the fluid is controlled byselecting an appropriate photoradiation source, an appropriate distanceof the photoradiation source from the fluid to be decontaminated, whichmay be increased through the use of light guides to carry thephotoradiation directly to the container for the fluid, an appropriatephotopermeable material for the container for the fluid, an appropriatedepth to allow full penetration of the photoradiation into thecontainer, photoradiation enhancers such as one or more additionalphotoradiation sources, preferably on the opposite side of the containerfrom the first, or reflectors to reflect light from the radiation sourceback into the container, appropriate flow rates for the fluid in thecontainer and an appropriate container length to allow sufficient timefor inactivation of microorganisms present. Temperature monitors andcontrollers may also be required to keep the fluid at optimaltemperature. FIG. 6 depicts a decontamination system of this inventionas part of an apparatus for separating blood components, and FIG. 7provides details of a preferred decontamination system.

For batch systems, it is preferred to place the fluid to bedecontaminated along with the photosensitizer in bags which arephotopermeable or at least sufficiently photopermeable to allowsufficient radiation to reach their contents to activate thephotosensitizer. Sufficient photosensitizer is added to each bag toprovide inactivation, preferably to provide a photosensitizerconcentration of at least about 10 μM, and the bag is agitated whileirradiating, preferably at about 1 to about 120 J/cm² for a period ofbetween about 6 and about 36 minutes to ensure exposure of substantiallyall the fluid to radiation. Preferably, a combination of visible lightand ultraviolet light is used concurrently. The photosensitizer may beadded in powdered form.

The method preferably uses endogenous photosensitizers, includingendogenous photosensitizers which function by interfering with nucleicacid replication. 7,8-dimethyl-10-ribityl isoalloxazine is the preferredphotosensitizer for use in this invention. The chemistry believed tooccur between 7,8-dimethyl-10-ribityl isoalloxazine and nucleic acidsdoes not proceed via singlet oxygen-dependent processes (i.e. Type IImechanism), but rather by direct sensitizer-substrate interactions (TypeI mechanisms). Cadet et al. (1983) J. Chem., 23:420-429, clearlydemonstrate the effects of 7,8-dimethyl-10-ribityl isoalloxazine are dueto non-singlet oxygen oxidation of guanosine residues. In addition,adenosine bases appear to be sensitive to the effects of7,8-dimethyl-10-ribityl isoalloxazine plus UV light. This is importantsince adenosine residues are relatively insensitive to singletoxygen-dependent processes. 7,8-dimethyl-10-ribityl isoalloxazineappears not to produce large quantities of singlet oxygen upon exposureto UV light, but rather exerts its effects through direct interactionswith substrate (e.g., nucleic acids) through electron transfer reactionswith excited state sensitizer species. Since indiscriminate damage tocells and proteins arises primarily from singlet oxygen sources, thismechanistic pathway for the action of 7,8-dimethyl-10-ribitylisoalloxazine allows greater selectivity in its action than is the casewith compounds such as psoralens which possess significant Type IIchemistry.

FIG. 7 depicts a stand-alone version of the decontamination assembly ofthis invention. The vaccine product containing infectious particles tobe inactivated (hereinafter referred to as the product) 180 is connectedto product line 186 which leads through pump 184 to decontaminationcuvette 164. Photosensitizer reservoir 166 is connected tophotosensitizer input line 168 equipped with input pump 170, and leadsinto product line 186 upstream from decontamination cuvette 164.Decontamination cuvette 164 is a photopermeable cuvette of a depth (d)and a length (I) selected to ensure decontamination. Cooling system 190combined with temperature monitor 192 are connected with decontaminationcuvette 164 for controlling the temperature of the fluid.Decontamination cuvette 164 is connected via light guide 162 tophotoradiation source 160. A photoradiation enhancer 163 is placedadjacent to (either touching or spaced apart from) decontaminationcuvette 164 to increase the amount of photoradiation reaching the bloodproduct in the cuvette. Decontaminated product line 188 leads fromdecontamination cuvette 164 to decontaminated product collection 182.

In operation, product 180 is conducted into product line 186 where it isjoined by photosensitizer from photosensitizer reservoir 166 flowing ata rate controlled by photosensitizer input pump 170 in photosensitizerinput line 68 which joins product line 186. The flow rate in productline 186 is controlled by pump 184 to a rate selected to ensuredecontamination in decontamination cuvette 164. Temperature monitor 192measures the temperature of fluid in cuvette 164 and controls coolingsystem 190 which keeps the temperature in the cuvette within a rangerequired for optimal operation. The product in decontamination cuvette164 is irradiated by photoradiation from photoradiation source 160conducted in light guide 162. The photoradiation source may comprise twoor more actual lights. The arrows indicate photoradiation from the endof light guide 162 propagating in the product inside transparentdecontamination cuvette 164. Adjacent to decontamination cuvette 164 isphotoradiation enhancer 163 which may be an additional source ofphotoradiation or a reflective surface. The arrows from photoradiationenhancer 163 pointing toward decontamination cuvette 164 indicatephotoradiation from photoradiation enhancer 163 shining on the productmaterial in cuvette 164. Decontaminated product exits decontaminationcuvette 164 via decontaminated product line 188 and is collected atdecontaminated product collection 182.

In one embodiment using 7,8-dimethyl-10-ribityl isoalloxazine from SigmaChemical Company as the photosensitizer, a light guide from EFOSCorporation, Williamsville, N.Y. composed of optical fibers is used. Thesystem is capable of delivering a focused light beam with an intensityof 6,200 mW/cm² in the region of 355-380 nm. It is also possible to useinterchangeable filters with the system to achieve outputs of 4,700mW/cm2 in the spectral region of 400-500 nm. In both cases, the outputof light in the region of 320 nm and lower is negligible. Light guidesof varying dimensions (3, 5 and 8 mm) are available with this system.The light exits the light guide tip with a 21 degree spread. The 8 mmlight guide is appropriate, correctly placed, to adequately illuminatethe face of the preferred decontamination cuvette which is a standardcuvette used on Cobe Spectra⁷ disposables sets from Industrial Plastics,Inc., Forest Grove, Oreg.

The flow rate is variable and is determined by the amount of lightenergy intended to be delivered to the sample. The flow rate iscontrolled by means of a peristaltic pump from the Cole-ParmerInstrument Company, Vernon Hills, Ill. Flow rates and type of inputstream may be controlled via a computer processor as is known to theart.

FIG. 22 depicts an embodiment of this invention in which fluid to bedecontaminated is placed in a bag 284 equipped with an inlet port 282,through which photosensitizer in powder form 284 is added from flask 286via pour spout 288. Shaker table 280 is activated to agitate the bag 284to dissolve photosensitizer 290 while photoradiation source 260 isactivated to irradiate the fluid and photosensitizer in bag 284.Alternatively, the bag can be provided prepackaged to containphotosensitizer and the fluid is thereafter added to the bag.

The methods of this invention do not require the use of enhancers suchas quenchers or oxygen scavengers, however these may be used to enhancethe process by reducing the extent of non-specific cell orprotein-damaging chemistry or enhancing the rate of pathogeninactivation. Further preferred methods using non-toxic endogenousphotosensitizers and endogenously-based derivative photosensitizers donot require removal of photosensitizers from the fluid afterphotoradiation. Test results show little or no damage to other bloodcomponents, e.g. platelets remain biologically active five dayspost-treatment.

EXAMPLES Example 1

Absorbance Profile of 7,8-dimethyl-10-ribityl isoalloxazine

A sample of 7,8-dimethyl-10-ribityl isoalloxazine (98% purity) wasobtained from Sigma Chemical Company. A portion of this sample wassubmitted for analysis using a scanning UV spectrophotometer. The rangestudied covered the region of 200 to 900 nm. For analysis, the samplewas dissolved in distilled water. A sample spectrum from this analysisis shown in FIG. 1.

Results were consistent with those reported in the literature for theabsorbance maxima and extinction coefficients for7,8-dimethyl-10-ribityl isoalloxazine Literature λmax (ε) Measured λmax(ε) 267 (32,359) 222 (30,965) 265 (33,159) 373 (10,471) 373 (10,568) 447(12,303) 445 (12,466)

Appropriate wavelengths for irradiation are 373 and 445 nm. Theextinction coefficients observed at these absorbance maxima issufficient to ensure adequate activation of the sensitizer in solution.

Example 2

Solubility of 7,8-dimethyl-10-ribityl isoalloxazine

Solubility in isolyte S, pH 7.4 Media

The maximum solubility of 7,8-dimethyl-10-ribityl isoalloxazine inIsolyte S media was determined as follows:

7,8-dimethyl-10-ribityl isoalloxazine was mixed with Isolyte S until aprecipitate was formed. The mixture was agitated at room temperature forone hour and vortex mixed to ensure complete dissolution of thesuspended material. Additional 7,8-dimethyl-10-ribityl isoalloxazine wasadded until a solid suspension remained despite additional vortexmixing. This suspension was then centrifuged to remove undissolvedmaterial. The supernatant from this preparation was removed and analyzedusing a spectrophotometer. The absorbance values of the solution weredetermined at 447 nm and 373 nm. From the extinction coefficients thatwere determined previously, it was possible to estimate theconcentration of the saturated solution

-   -   Concentration (373)=110 μM=42 μg/mL    -   Concentration (447)=109 μM=40.9 μg/mL        Solubility in ACD-A Anticoagulant

The same procedure described above was repeated using ACD-AAnticoagulant. The values obtained from these measurements were asfollows:

-   -   Concentration (373)=166 μM=63 μg/mL    -   Concentration (447)=160 μM=60.3 μg/mL

The values obtained from these studies indicate an upper limit ofsolubility of the compound that may be expected.

Example 3

Photodecomposition of 7,8-dimethyl-10-ribityl isoalloxazine in AgueousMedia

A solution of 7,8-dimethyl-10-ribityl isoalloxazine in Sigma ACD-A wasprepared at a concentration of 63 μg/mL. This preparation was taken upinto a glass pipette and placed in the path of a UV light source (365 nmλmax with filters to remove light below 320 nm). The suspension wasirradiated for specific intervals at which aliquots were removed forspectroscopic analysis. The absorbance of the dissolved7,8-dimethyl-10-ribityl isoalloxazine was monitored at 373 and 447 nm ateach time interval. The results are depicted in FIG. 3 and Table 1.TABLE 1 Photodecomposition of 7,8-dimethyl-10-ribityl isoalloxazine UponExposure to UV Light (365 nm) in Acid Solution Irradiation Time % ofInitial, 373 nm % of Initial, 447 nm 0 100 100 5 87.3 61.6 10 90.5 76.615 100 70

The absorption profile for the solution at 373 nm indicates that nosignificant decomposition of the reagent occurred over the entireirradiation period. The absorbance of light at this wavelengthcorresponds to n-π* electronic transitions. The absence of a decrease inthe intensity of this peak over time indicates that the ring structureof the molecule is intact despite prolonged irradiation under theseconditions. The absorbance of the molecule at 447 nm is due to π-π*electronic state transitions. The decrease in the absorbance of themolecule at this wavelength with increasing irradiation times isindicative of subtle alterations in the resonance structure of themolecule. This change is most likely due to the loss of ribose from thering structure of the 7,8-dimethyl isoalloxazine backbone and theformation of 1,8-dimethylalloxozine as a result. These changes areconsistent with literature reports on the behavior of the molecule uponirradiation with UV light.

The apparent lack of decomposition of the ring structure of the moleculeis in stark contrast to observations with psoralen-based compounds undersimilar conditions. During irradiation, a significant fluorescence ofthe molecule in solution was observed. This behavior of the molecule isconsistent with the resonance features of the ring structure andprovides a means for the dissipation of energy in the excited statemolecule in a non-destructive fashion.

Example 4

Flow System Concept Evaluation

Light Transmission Properties of Existing Spectra Cuvette

The existing Spectra cuvette is composed of polycarbonate. The lighttransmission properties of this cuvette were measured at 373 and 447 nmby placing the cuvette in the light path of a UV spectrophotometer. Thevalues obtained were as follows: Wavelength of Light % Transmittance 373nm 66% 447 nm 80%

These results are consistent with those reported in the literature forpolycarbonate plastics (see FIG. 4). The literature values indicate asteep shoulder for the transmission of light through polycarbonates inthe region of 300 nm. For the region above 350 nm, the lighttransmission properties are adequate for this application.

Light Flux Requirements Calculated as a Function of Flow Rates

In order for a flow system to be feasible, the sample must be providedwith an adequate flux of light during its presence in the beam path. Ifthe proposed Spectra cuvette were to serve this purpose, then it ispossible to estimate the light flux requirements as a function of flowrates through the cuvette as follows:

The volume of solution present in the irradiation zone of the cuvette isca. 0.375 mls. The transit time for a cell in this region of the cuvettecan be determined from the following equation:$T = \frac{{Volume}\quad{of}\quad{Cuvette}\quad({mis})}{{Flow}\quad{Rate}\quad\left( {{mls}/\min} \right)}$

At 100 mls per minute, the transit time (T) would be 0.00375 min=0.225seconds.

The energy to which a sample is exposed is dependent on the fluxaccording to the following equation:${{Energy}\quad\left( {E,{{Joules}/{cm}^{2}}} \right)} = \frac{{{Flux}{\quad\quad}\left( {\varphi,{{mW}/{cm}^{2}}} \right)}*{Time}\quad\left( {T,\sec} \right)}{1000}$

If we assume that 1 Joule/cm² is required to activate the sensitizeradequately and the transit time (T) is 0.22 seconds (i.e., flow rate of100 mls/min through the cuvette), then the required Flux during thesample=s transit through the cuvette is 4,545 mW/cm². A graph depictingthe relationship of the required flux from the light source to flowrates through the cuvette is provided in FIG. 5.

These results indicate that, for a flow system to operate properly, UVsources with outputs in the region of Watts/cm² are required.

FIG. 2 shows how absorbance should vary with concentration of platelets.

Example 6

Effects of Virus Inactivation Treatment on Platelet In Vitro Parameters

Effects of virus inactivation treatment on platelet in vitro parameterswere evaluated. Platelet preparations were treated with7,8-dimethyl-10-ribityl isoalloxazine in combination with UV light.Various in vitro parameters were used as monitors of platelet functionin order to determine the extent of changes induced by the treatmentconditions. Factors such as energy level of UV light exposure, dose of7,8-dimethyl-10-ribityl isoalloxazine used, and sample processingconditions were examined for their impact on platelet qualitypost-treatment. Results from this study are used to establish anappropriate treatment window for inactivation of HIV-1 withoutcompromising platelet function.

Samples were prepared with three different concentrations of7,8-dimethyl-10-ribityl isoalloxazine. Platelets obtained from astandard Spectra LRS collection were used for these studies.

Starting samples were centrifuged to concentrate the platelet pellet.The pellet was resuspended in a 70:30 (Isolyte S, pH 7.4; McGaw, Inc.Media:Plasma) solution. 7,8-dimethyl-10-ribityl isoalloxazine at thespecified concentration, was present in the plasma:media mixture. Theplatelet suspension was then passed through a UV irradiation chamber atone of three specified flow rates. The flow rates were directlycorrelated to the energy level of exposure for the cells/media mixturewhich passes through the irradiation chamber. After flowing through theirradiation chamber, samples were stored in a citrate plasticizedsampler bag for subsequent analysis.

Following irradiation, in vitro measurements of platelet function,including hypotonic shock response (HSR), GMP-140 expression, pH, pCO₂,pO₂, platelet swirl, and cell count, were evaluated in order todetermine the effects of the treatment protocol on cell quality.

Platelet quality was monitored as a function of irradiation conditions(sensitizer concentration and flow rates/Energy levels). The plateletquality includes parameters such as HSR response, GMP-140 activation,etc. The flow rates that are studied can be related to the Energy ofexposure as follows: $\begin{matrix}{{{Transit}\quad{Time}\quad\left( {T,\sec} \right)} = {{Exposure}\quad{Time}}} \\{= \frac{0.375\quad{mls}}{\left( {F_{r}/60} \right)}}\end{matrix}$ F_(r) = Flow  Rate  (mls/min )0.375  mls = Cuvette  Volume  (mls)${T\quad\left( \sec \right)} = \frac{22}{F_{r}}$${{Energy}\quad\left( {{Joules}/{cm}^{2}} \right)} = \frac{{Flux}\quad\left( {\varphi,{{mW}/{cm}^{2}}} \right)*T\quad\left( \sec \right)}{1000}$$E = \frac{\varphi*0.022}{F_{r}}$

The effect of energy of UV exposure and concentration of7,8-dimethyl-10-ribityl isoalloxazine on the stability and viability oftreated platelets was evaluated. Three energy levels and threeconcentration levels were evaluated as follows: Energy Levels: 1, 5, 9J/cm²* 7,8-dimethyl-10-ribityl isoalloxazine 1, 50, 100 μM**Concentrations:*Levels of total energy exposure were determined by the flow rate of thesuspension through the irradiation chamber in accordance with theconversion chart of Table 4.**Since the media is diluted 70:30 (Media:Plasma) the stockconcentration of 7,8-dimethyl-10-ribityl isoalloxazine in media aloneprior to mixing with the plasma was adjusted appropriately. Thisrequired starting concentrations in Isolyte S of 1.43, 71.4, and 143 μM.

TABLE 4 Energy Exposure Levels as a Function of Flow Rate Through theIrradiation Chamber Time to process 20 mls Energy Delivered (J/cm²) FlowRate (mls/min) (minutes) 1 16.90 1.18 2 8.45 2.37 3 5.83 3.55 4 4.224.73 5 3.38 5.92 6 2.82 7.10 7 2.41 8.29 8 2.11 9.47 9 1.88 10.65 101.69 11.84Flux = 3640 mW/cm²; chamber volume = 0.117 mls.Values for treated samples were compared to control groups. The controlsamples included the following:Untreated Sample in Plasma (Historical Control)+Flow-UV-7,8-dimethyl-10-ribityl isoalloxazineProcedure

A normal donor platelet apheresis product was obtained from an AABBacceredited blood banking facility. The sample was collected usingstandard Spectra LRSA procedures. All manipulations or proceduresdescribed below were performed with standard laboratory safetyprocedures and methods. The unit number and blood type were recorded.All samples were used within 24 hours of collection. Aseptic procedurewas followed for all sample transfers and processing steps.

The sample was transferred to a 500 mls PVC transfer pack andcentrifuged at 5000×g for five minutes to pack the platelets. Plasma wasthen removed from the platelet pellet using a standard plasma press. Theplasma was retained for further use. The plasma removed from the cellpellet was then mixed with a stock solution of Isolyte S, pH 7.4; McGaw,Inc. This stock solution of media was prepared by adding apre-determined amount of 7,8-dimethyl-10-ribityl isoalloxazine toIsolyte S to provide final concentrations of 1.43, 71.4, and 143 μM.Following addition of 7,8-dimethyl-10-ribityl isoalloxazine the stocksolution was filtered through a 0.22 μM sterile filter. The stocksolution was then mixed with autologous plasma in a 70:30 (v:v) ratio toprovide final 7,8-dimethyl-10-ribityl isoalloxazine concentrations of 1,50, and 100 μM respectively. During preparation of the7,8-dimethyl-10-ribityl isoalloxazine stock solutions, care was taken toavoid exposure to light. Samples were prepared according as follows:  1μM 2 samples 100 μM 2 samples  50 μM 1 sample 

The platelet pellet was then resuspended in the plasma:media mixture tothe original volume of the starting sample. The sample was connected toa flow apparatus comprising a container for cells and photosensitizer, acontainer for media, said containers being connected via valved lines toa single line for mixed cells/sensitizer and media equipped with a pump.Mixed cells/sensitizer and media were flowed into a cuvette held in aholder with a mirrored wall, irradiated by a light source. Thisirradiation chamber was equipped with a temperature probe. After passingthrough the cuvette, fluid was collected in a product bag.

The tubing set was initially primed with Isolyte S media. Five minutesprior to the start of the test sample flow, the light source wasactivated. Temperature was monitored during this interval and kept lowerthan 32° C. in the irradiation chamber.

The flow rate for the sample through the irradiation chamber wasdetermined by the chart of Table 4. Flow rates which provide totalirradiation energy levels of 1, 5 and 9 J/cm² were utilized according tothe following testing matrix:

-   Sample Run #1: 7,8-dimethyl-10-ribityl isoalloxazine Concentration=1    μM

A. +7,8-dimethyl-10-ribityl isoalloxazine+1 J/cm²

B. +7,8-dimethyl-10-ribityl isoalloxazine+9 J/cm²

-   Sample Run #2: 7,8-dimethyl-10-ribityl isoalloxazine=100 μM

A. +7,8-dimethyl-10-ribityl isoalloxazine+1 J/cm²

B. +7,8-dimethyl-10-ribityl isoalloxazine+9 J/cm²

-   Sample Run #3: 7,8-dimethyl-10-ribityl isoalloxazine=50 μM

A. +7,8-dimethyl-10-ribityl isoalloxazine+5 J/cm²

-   Sample Run #4: Control Sample, 7,8-dimethyl-10-ribityl    isoalloxazine=0 μM

A. +Flow-UV-7,8-dimethyl-10-ribityl isoalloxazine

All samples were identified by the run number and sample letterdesignation corresponding to treatment condition (i.e., 1A). Each sampleset was run for a total of 2 replicates. The order in which samples weretreated was determined by assignment according to a random numbergenerator.

A sample volume of 20 mls per run condition was collected for eachsample. These samples were collected into citrate plasticized samplingbags (53 mls total volume) and stored for analysis. The temperature ofthe sample and the irradiation chamber was noted at the start,mid-point, and end of each run.

An initial aliquot from each preparation was removed post-treatment foranalysis. Parameters for analysis included cell count, pH, pCO2, pO2,platelet swirl, HSR, and GMP-140 analysis. The remaining portion of thesample was placed in an end-over-end platelet agitator in a +22incubator and stored for five days post-treatment. On day 5, a secondaliquot was removed and analyzed for the same in vitro parameters.

The following equipment was used: Nikon Labophot microscope;Serono-Baker System 9000 Hematology Analyzer; analytical balance;platelet incubator (+22 Celsius) and rotator; laboratory refrigerator(+4 Celsius); Mistral 3000i Centrifuge; Corning Blood Gas Analyzer;Becton-Dickinson FACSCALIBUR Flow Cytometer; UV irradiation chamber; UVradiometer (UVX Radiometer, UVP, Inc.); EFOS Ultracure 100SS Plus (365nm maximum output and 340 nm bandpass filters); and temperature probe(thermocouple).

Results for each set of test variables were compared for the definedconditions of energy of exposure and concentration of7,8-dimethyl-10-ribityl isoalloxazine. Direct comparison to theuntreated control sample was made and significant differences defined bya probability p>0.05 from a paired, one-tailed, Student's T-Testanalysis.

The results from these studies were summarized as follows:

-   01 At sensitizer concentrations in excess of 10 μM and platelet    concentrations above 1.5E+06/μL, there was a drop in sample pH by    day 2. The pH declined steadily beyond day 2 of storage reaching    unacceptable levels (<6.5) by day 3 of storage. All other in vitro    parameters followed the pattern observed with sample pH.-   02 This decrease in sample pH occurred regardless of whether or not    the sample was exposed to UV light.-   03 At platelet concentrations of 5.4E+05/μL, there was no drop in    sample pH after extended storage at any sensitizer concentration    studied up to 100 μM.

At sensitizer concentrations up to 10 μM, platelet concentrations above1.5E+06/μL, and UVA levels up to 10 J/cm2, measured platelet propertieswere comparable to control, untreated cells. These remained comparableto control levels after five or more days of storage post-treatment.

These studies on platelet function post-treatment provided a clearwindow in which cell properties were maintained at levels comparable tountreated cells. The results also indicated that by varying the storageor treatment conditions for the cells this window can be expanded. Theobserved effect of 7,8-dimethyl-10-ribityl isoalloxazine with or withoutUV light on sample pH suggests a metabolic effect of this additive whichmay be moderated by changes in the storage or processing conditions ofthe samples.

Example 7

Measurements of Shear Stresses on Red Cells As a Function of Flow Rateand Sample Hematocrit

The low levels of UV light penetration into red cell samples at highhematocrits raised the need to understand the effects of passing redcells through narrow openings in the light path. Reduction in samplethickness in the light path should increase delivery of UV dose at highsample hematocrits. In order to confirm this approach, several pressuredrop measurements were undertaken using openings of varying dimensions.A pressure gauge was placed in line with a peristaltic pump bothupstream and downstream from the narrowed openings. Whole blood ofvarying hematocrits was passed through the openings at controlled flowrates. Differences in the pressure readings at both locations permitteddirect measurement of the pressure drop across the opening. Using thisvalue and the dimensions of the opening, it was possible to determinethe shear stress experienced by the red cells as they passed through thenarrowed cell using the following equation:${\Delta\quad P} = {\frac{8{\mu L}\quad Q}{{gd}^{3}w}\quad{Pressure}\quad{Drop}}$$\Gamma_{w} = {\frac{4\quad\mu\quad Q}{{gwd}^{2}}\quad{Shear}\quad{Stress}}$For blood,

μ=Viscosity=0.0125/(1-Hematocrit)

g=gravitational constant=981

Q=Flow Rate=mis/sec

I, w, d=Dimensions of opening in cm TABLE 5 Measurement of Shear Stresson Red Cells As Functions of Flow Rate and Sample Hematocrit DpmeasDpmeas Dpmeas 0.08 × 0.008 (dynes/cm²) 0.08 × 0010 (dynes/cm²) 0.08 ×0.012 (dynes/cm²) 41% Q = 3.38 1.5 95.9 1.0 77.6 0.0 0.0 HCT 64% Q =3.38 4.0 255.8 3.0 232.9 2.0 182.1 HCT 41% Q = 16.9 9.7 618.4 7.0 543.44.7 425.3 HCT 61% Q = 16.9 20.7 1321.9 12.3 957.2 8.7 789.6 HCT DpmeasDpmeas Dpmeas 0.10 × 0.008 (dynes/cm²) 0.1 × 0.010 (dynes/cm²) 0.1 ×0.012 (dynes/cm²) 41% Q = 3.38 2.0 93.7 1.0 60.3 1.0 73.5 HCT 64% Q =3.38 4.5 210.8 3.0 180.9 2.0 146.9 HCT 41% Q = 16.9 12.7 593.6 7.0 422.14.7 343.0 HCT 61% Q = 16.9 23.3 1093.0 14.7 884.6 12.0 881.4 HCT DpmeasDpmeas Dpmeas 0.15 × 0.008 (dynes/cm²) 0.15 × 0.010 (dynes/cm²) 0.15 ×0.012 (dynes/cm²) 41% Q = 3.38 3.0 97.4 1.2 49.2 1.0 49.0 HCT 64% Q =3.38 6.5 211.0 3.5 143.5 2.0 97.9 HCT 41% Q = 16.9 15.3 497.7 8.3 341.65.7 277.6 HCT 61% Q = 16.9 35.7 1158.1 18.0 738.1 12.7 620.4 HCT

In previous experiments, it was determined that shear stresses of1,000-2,000 dynes/cm2 for intervals of 1 -10 minutes or levels of5,000-7,000 dynes/cm² for intervals of approximately 10 msec weresufficient to induce red cell hemolysis. Only in the case of the highestsample hematocrit (61 %) and highest flow rate (16.9) did values exceed1,000 dynes/cm². This occurred only for openings of the narrowest width(0.008 inches).

Values for the light penetration depth using the proposed configurationindicate that delivery in sufficient UV energy to drive virusinactivation processes is achievable even for samples with highhematocrits.

Results from shear stress analysis on red cell samples subjected to flowindicate that flow path dimensions may be significantly reduced and highflow rates maintained without risking red cell hemolysis.

Example 8

A platelet concentrate was mixed with the platelet additive solutionIsolyte S at a ratio of 20:80 platelet concentrate:lsolyte S. Mixturesof platelet concentrates and platelet additive solutions are referred toherein as in “media.” Platelet concentrate without additive solution isreferred to herein as in “plasma.” Both were spiked with Listeriamonocytogenes. Vitamin K5 was then added to each in the amount of 300pg/mL B. Each was then exposed to UV, visible or room light in thecuvette apparatus of FIG. 6 with the results shown in Table 6. TABLE 6Log Inactivation (cfu/mL) K5 in Media K5 in Plasma UV, 40 J/cm² 4.2 Logs0.1 Logs VIS, 40 J/cm² 4.2 Logs 0.1 Logs Room Light   0 Logs   0 LogsUV Light = 365 nmVIS Light = 419 nmPathogen = Listeria monocytogenesConcentration of K5 = 300 μg/mL

Example 9

Media and plasma as described above containing vitamin K5 were spikedwith bacteria and irradiated or exposed to room light only (K5-light) asshown in Table 7, and growth evaluated after three days of incubation.Inactivation of some species was seen in the absence of irradiation.TABLE 7 Plasma Spike Level Media K5 − (cfu/mL) K5 − Light K5 + LightLight P. aeruginosa 3.4 Logs − − − − S. aureus 2.1 Logs − − + + S.epidermidis 3.2 Logs − + − − L. monocytogenes 3.5 Logs − − + + E. coli3.1 Logs − − + −UV Light = 365 nm, 40 J/cm²+ = Growth detected after three days incubation− = No Growth detected after three days incubationConcentration of K5 = 300 μg/mL

Example 10

Media made using a platelet concentrate as described in Example 8 andIsolyte S at a ratio of Isolyte S:platelet concentrate of 70:30 andcontaining 300 pg/mL vitamin K5 was spiked with several species ofbacteria and irradiated at energy levels of 30 and 60 J/cm².Inactivation as a function of energy of irradiation is set forth inTable 8 and FIG. 7. TABLE 8 Energy (J/cm²) S. aureus S. epidermidis L.monocytogenes E. coli 0 4.3 2.6 2.8 3.5 30 3.6 2.7 2 2 60 3.2 2.5 1 1

Example 11

To platelet concentrate as described in Example 8 and to 70:30 media asdescribed in Example 10 was added 10 μM of 7,8-dimethyl-10-ribitylisoalloxazine. The platelet concentrate and media were spiked with S.aureus or S. epidermidis, and irradiated at 80 J/cm² and 30 J/cm² andinactivation measured as above. Results are shown in FIG. 8.

Example 12

To plasma concentrate as described in Example 8 contained in a standardblood bag was added 25 μM 7,8-dimethyl-10-ribityl isoalloxazine inpowder form. The bag was spiked with bacteria as shown in Table 9,agitated and exposed to 120 J/cm² radiation. Inactivation results areset forth in Table 9. TABLE 9 Pathogen Log Inactivation (cfu/mL) S.aureus 1.7 Logs S. epidermidis 3.5 Logs P. aeruginosa 3.6 Logs E. coli4.1 Logs

Example 13

To platelet concentrate as described in Example 8 was added7,8-dimethyl-10-ribityl isoalloxazine, alloxazine mononucleotide, or7-8-dimethyl alloxazine, followed by spiking with S. aureus or S.epidermidis, and irradiation at 80 J/cm². Inactivation results are shownin Table 10. TABLE 10 Log Inactivation (cfu/mL) StaphylococcusStaphylococcus aureus epidermidis 7,8-dimethyl-10-ribityl isoalloxazine,1.9 Logs 4.1 Logs 10 μM alloxazine mononucleotide, 10 μM 1.6 Logs 5.6Logs 7-8-dimethyl alloxazine, 7 μM 1.6 Logs 2.9 Logs

Example 14

To platelet concentrate of Example 8 was added 10 μM7,8-dimethyl-10-ribityl-isoalloxazine. Aliquots contained no additive,10 mM ascorbate or 10 mM Kl as a Aquencher@ or antioxidant. Thesolutions were spiked with HSV-2, φX174, S. epidermidis or S. aureus andirradiated at 80 J/cm². Results are shown in FIG. 9.

Example 15

To platelet concentrates of Example 8 were added varying concentrationsof 7,8-dimethyl-10-ribityl-isoalloxazine. These solutions were spikedwith herpes simplex virus type II (HSV-I), a double-stranded DNAenvelope virus. Irradiation was done at 80 J/cm². The experiment wasreplicated three times. In all three trials complete inactivation wasachieved. Results are shown in FIG. 10.

Example 16

The protocol of Example 15 was followed using S. epidermidis instead ofHSV II at energies of irradiation of 40, 80 and 120 J/cm². Inactivationresults are shown in FIG. 11.

Example 17

The protocol of Example 15 was followed using φX174, a single strandedDNA bacteriophage, at varying concentrations of7,8-dimethyl-10-ribityl-isoalloxazine and energies of irradiation.Inactivation results are shown in FIG. 12.

Example 18

To platelet concentrates of Example 8 was added 10 μM7,8-dimethyl-10-ribityl-isoalloxazine. These were spiked with S. aureusor φX174 and irradiated at varying energies of irradiation with a 50:50mixture of visible and ultraviolet light. Inactivation results are shownin FIG. 13.

Example 19

The protocol of Example 18 was followed using S. epidermidis and HSV-IIas the microorganisms. A 50:50 mixture of ultraviolet and visible lightwas supplied by DYMAX light source. Inactivation results are shown inFIG. 14.

Example 20

To platelet concentrate of Example 8 was added 10 μM7,8-dimethyl-10-ribityl-isoalloxazine in powdered form. Tests with andwithout added ascorbate were conducted. 150 ml of the test solutionswere placed in a Spectra blood bag and shaken and exposed to varyingenergies of irradiation using 50:50 visible:ultraviolet light. Afterreceiving 40 J/cm², the contents of each bag were transferred to a newbag to avoid errors due to microorganisms which may have remained in thespike port of the bag. Inactivation results are shown in FIG. 15.Downward arrows indicate inactivation to the level it was possible todetect (2.5 log titre).

Example 21

To platelet concentrate of Example 8 and platelet concentrate in IsolyteS at 30:70 platelet concentrate:lsolyte S, was added 20 μM7,8-dimethyl-10-ribityl-isoalloxazine. These were spiked with vacciniavirus, a double stranded DNA envelope virus, and exposed to 60 J/cm²visible light or mixed (50:50) visible and ultraviolet light using aDYMAX 2000 UV light source for 30 minutes. The limit of detection was1.5 logs. Inactivation results are shown in FIG. 16. Comparisons weredone using no photosensitizer, photosensitizer in Isolyte S media alone,platelets in Isolyte S media, platelets in Isolyte S media using8-methoxy psoralen instead of 7,8-dimethyl-10-ribityl-isoalloxazine, andplatelet concentrate in Isolyte media (30:70).

Example 22

Samples of platelet concentrate in Isolyte S media 30:70, with andwithout 10 μM 7,8-dimethyl-10-ribityl-isoalloxazine were spiked withvaccinia virus and irradiated at 60 J/cm² with 50:50 visible:UV lightfor varying periods of time and inactivation results compared as shownin FIG. 17.

Example 23

To samples of platelet concentrate as described in Example 8 were added5 μM or 50 μM 7,8-dimethyl-10-ribityl-isoalloxazine. Samples were spikedwith HIV 1. Using the cuvette flow cell shown in FIG. 6, samples wereirradiated with 50:50 visible:UV light at varying energies using an EFOSlight system. Inactivation results are show in FIG. 18.

Example 24

HIV-infected ACH-2 cells were added to samples of platelet concentratedescribed in Example 8.5 or 50 μM of7,8-dimethyl-10-ribityl-isoalloxazine were added to the samples. Theprotocol of Example 23 was followed, and inactivation results are shownin FIG. 19. The presence of HIV was assayed by its cytopathic effect ontest cells.

Example 25

The protocol of Example 24 was followed and the presence of HIV assayedby quantifying the level of p24 antigen production. Inactivation resultsare show in FIG. 20.

Example 26

To samples of platelet concentrate as described in Example 8 and mediacontaining 30% platelet concentrate and 70% PASIII™ media were added 6mM ascorbate and 14 μM 7,8-dimethyl-10-ribityl-isoalloxazine. Sampleswere spiked with HSV-II. Inactivation results are show in FIG. 21 andTable 11. TABLE 11 Energy 30:70 Energy 90:10 Time (UV + VIS) PC:Media(UV + VIS) PC:Media (Minutes) J/cm² log virus titre J/cm² log virustitre 0 0 5.6 0 5.6 1.5 5 2.5 40 3.3 3 10 2.5 80 1.5 No Detectable Virus4.5 15 2.3 120 1.5 No Detectable Virus 6 20 1.8 9 30 1.6 12 40 24 80 36120

It will be readily understood by those skilled in the art that theforegoing dewcription has been for purposes of illustration only andthat a number of changes may be made without departing from the scope ofthe invention. For example, other photosensitizers than those mentionedmay be used, preferably photosensitizers which bind to nucleic acid andthereby keep it from replicating, and more preferably those which arenot toxic and do not have toxic breakdown products. In addition,equivalent structures to those described herein for constructing aflow-through system for decontamination of fluids using photosensitizersmay be readily devised without undue experimentation by those skilled inthe art following the teachings hereof.

1. A method of making a vaccine for preventing the replication of livetumor cells in a mammal comprising the steps of: removing a tumor fromthe mammal; purifying at least some of the tumor cells from the tumor;inactivating the tumor cells by exposing the tumor cells to anendogenous photosensitizer and light at a sufficient wavelength toprevent replication of the tumor cells but not substantially destroy theantigenic determinants of the tumor cells; and suspending theinactivated tumor cells in a suspension solution.
 2. The method of claim1 wherein the suspension solution is chosen from the group consistingof: saline and water.
 3. The method of claim 1 wherein the suspensionsolution is chosen from the group consisting of an antibiotic and apreservative.
 4. The method of claim 1 wherein the suspension solutionfurther comprises an adjuvant.
 5. The method of claim 1 wherein thephotosensitizer is 7,8-dimethyl-10-ribityl isoalloxazine.
 6. A methodfor producing immunity in a mammal comprising; administering to themammal a vaccine produced by the method of claim 1; and allowing theimmune system of the mammal to mount an immune response to the vaccine.7. A method of treating a vaccine to inactivate microorganisms which maybe present therein comprising the steps of: a. mixing an effectivenon-toxic amount of an endogenous photosensitizer or endogenously-basedderivative photosensitizer with the vaccine; b. exposing the vaccine tolight of a sufficient wavelength to activate the photosensistizer;whereby at least some of the microorganisms are inactivated.
 8. Themethod of claim 7 wherein the endogenous photosensitizer is selectedfrom the group consisting of: riboflavin, lumiflavin, lumichrome,napthquinones, napthels and napthalenes.